Trypanosomal variant surface glycoprotein expression in human 1 African trypanosomiasis patients 2 3

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Introduction
combination of native LiTat1.3 and another VSG, LiTat1.5 [27,28], or the combination of 119 a VSG with the invariant surface glycoprotein ISG 65 [29]. Currently, there is no 120 serological test for diagnosis of infection with T. b. rhodesiense.  [36]. For technical reasons, this study relied on RNA isolated from parasites passaged 147 through small animals after collection from the natural host. As VSG expression may 148 change during passage, the data obtained from these samples is somewhat difficult to 149 interpret. To better understand the characteristics of antigenic variation in natural T. 150 brucei infections, we sought to analyze VSG expression in T. brucei field isolates from 151 which RNA was directly extracted.

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In the present study, we used VSG-seq to determine the number and diversity of VSGs   For minors, additional written consent was obtained from their legal representative.   The cerebrospinal fluid was examined for white blood cell count and the presence of 193 trypanosomes to determine the disease stage and select the appropriate treatment.

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Patients were questioned about their place of residence. accession numbers PRJEB27207 and PRJEB18523), we processed raw reads using 253 the VSG-seq pipeline available at https://github.com/mugnierlab/VSGSeqPipeline. 254 Briefly, VSG transcripts were assembled de novo from quality-and adapter-trimmed 255 reads for each sample (patient or patient replicate) from raw reads using Trinity (version  Two T. b. gambiense patient VSGs (Patients 11 and 13) showed likely assembly errors.

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In one case, a VSG was duplicated and concatenated, and in another, two VSGs were 268 concatenated. These reference files were manually corrected (removing the duplicate or  For T. b. rhodesiense, we aligned each patient's data to its own VSG ORF assembly.

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RPKM values for each VSG in each sample were generated using MULTo (

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To investigate VSG expression in natural human infections, we performed VSG-seq on 390 RNA extracted from whole blood collected from 12 human African trypanosomiasis 391 patients from five locations in the Kwilu province of DR Congo ( Figure 1A). We  (Table 1). Using RNA extracted from 2.5 mL of whole blood from each patient, we 395 prepared libraries for VSG-seq in three separate batches for each technical replicate. 396 We amplified T. brucei RNA from host/parasite total RNA using a primer against the T.      gambiense VSGs; Figure 2A).  sequences across all blood and CSF samples ( Figure 3A, Supplemental Figure 4).

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SRA, the VSG-like protein that confers human serum resistance in T. b. rhodesiense 513 [60], was expressed in all patient samples.

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The HMM pipeline determined types for 74 of these VSG sequences; the remaining 516 sequences appeared to be incompletely assembled, presumably due to insufficient read  Another source for bias in expressed VSG type is the composition of the genomic VSG 572 repertoire. We could directly compare the genomic and expressed VSG repertoire for 573 EATRO1125 mouse infections, as the 'VSGnome' for this strain has been sequenced.